Hepatitis C virus (HCV) infection is the most significant risk factor for the development of hepatocellular carcinoma (HCC), a major liver malignancy and cancer type worldwide. The molecular genetics and signaling pathways associated with HCV-associated HCC pathogenesis remain poorly understood. Our long-term goal is to elucidate the molecular mechanisms underlying progression of chronic HCV to HCC. Autotaxin (ATX), an ectonucleotide pyrophosphatase/phosphodiesterase 2 enzyme and a potent cell stimulating motogen is over-expressed in various malignant tumor tissues and has been implicated to confer the tumorigenic and metastatic potential of a variety of cancer cells. In 2007, two groups provided the first evidence that the serum ATX activity and plasma lysophosphatidic acid (LPA) levels are increased in chronic hepatitis C in association with liver fibrosis, and that ATX and genes related to ATX signaling pathway were upregulated in HCC patients co-infected with HCV. Is this the cause or consequence of HCV infection? And how does aberrant regulation of ATX impact HCV replication and pathogenesis? We postulate that "ATX over-expression and signaling may be an important determinant in the replication and pathogenesis of HCV". Our hypothesis is based on some novel and vital clues linking ATX expression to HCV infection and pathogenesis. In preliminary experiments we observed that ATX mRNA expression is upregulated in the liver tissues of HCV-infected patients and its expression is further enhanced in HCV- associated HCC samples, consistent with the 2007 clinical study. Further, the expression levels of ATX mRNA were upregulated in HCV replicon cells versus replicon cured cells, suggesting a role for HCV in inducing ATX expression. This observation coupled with our data that HCV replicon cells secrete ATX, suggest that the HCV infected liver may potentially contribute to the aberrant expression of ATX in HCV- infected patients and patients with HCV-associated HCC. We found that the ATX promoter was activated and stimulated in HCV replicon cells, thus implicating HCV proteins and host-factors in HCV-infected cells to potentially play a role in aberrant regulation of ATX. Further, over-expression of ATX in HCV RNA bearing human hepatoma cells induced RhoA expression, activated RhoA and stimulated HCV RNA replication. Conversely, blockage of ATX secretion abrogated RhoA induction and ablated HCV RNA replication. We envision that ATX/LPA over-expression during the course of HCV infection may de-regulate mechanisms involved in Rho GTPase signaling thus contributing towards HCV pathogenesis. To test our hypothesis and to extend our findings two independent but complementary specific aims are proposed. In Aim 1 we will investigate the role of viral proteins and host factors in aberrant regulation of ATX expression during HCV infection and pathogenesis. In Aim 2, we propose to address the role of ATX in HCV replication and pathogenesis mechanisms. To investigate these aspects, we propose to employ nontransformed human hepatocytes (HH4 cells) conditionally expressing the full-length HCV ORF (HH4-HCV) as models for the early stage of HCV infection, and C-5B replicon cells which replicate HCV RNA in hepatoma cells to address questions pertaining to the scenario in the HCV infected tumor microenvironment. These objectives will be achieved via multi-prong approach, including cell culture, biochemical and molecular genetics. We envisage that our findings will provide crucial insights into modulation of ATX during HCV infection and further clarify the role of ATX in HCV replication and pathogenesis. These investigations may provide rational target for therapeutic intervention of HCV. PUBLIC HEALTH RELEVANCE: The proposed studies have the potential to provide crucial insights into regulation and role of autotaxin (ATX), a tumorigenic and metastatic protein, in HCV infection and pathogenesis mechanisms and may likely identify novel modalities for therapeutic intervention of Hepatitis C.